Chemical Profile of Leaves and Roots of Miracle Fruit (Synsepalum Dulcificum)

American Chemical Science Journal 12(1): 1-8, 2016, Article no.ACSJ.20456 ISSN: 2249-0205

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Chemical Profile of Leaves and Roots of Miracle Fruit (Synsepalum dulcificum)

V. N. Osabor 1*, R. A. Etiuma 1 and M. U. Ntinya 1

1Department of Pure and Applied Chemistry, University of Calabar, Nigeria.

Authors’ contributions

This work was carried out in collaboration between all authors. Author VNO designed the study, managed the literature searches and performed the statistical analysis. Author RAE wrote the protocol and wrote the first draft of the manuscript. Author MUN managed the laboratory analyses of the study. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/ACSJ/2016/20456 Editor(s): (1) Silvia Antonia Brandán, Inorganic Chemistry Institute, National University of Tucumán (UNT), Argentina. Reviewers: (1) Anthony Cemaluk Egbuonu, Michael Okpara University of Agriculture, Umudike, Nigeria. (2) Rajendra Nath, King George’s Medical University, Lucknow, India. (3) Bhaskar Sharma, Suresh Gyan Vihar University, Rajasthan, India. Complete Peer review History: http://sciencedomain.org/review-history/12772

Received 28 th July 2015 Accepted 24 th August 2015 Original Research Article nd Published 22 December 2015

ABSTRACT

Proximate analysis, elemental analysis and phytochemical analysis were conducted on leaves and roots of S. dulcificum (miracle fruit). The results of proximate analysis of the leaves sample showed the following compositions: protein (6.62 ±0.02)%, crude lipid (12 ±2.00)%, crude fibre (17.5 ±0.50)%, moisture (40.30 ±1.53)%, ash (6.70 ±2.00)% and carbohydrate (57.60 ±0.01)%, while that of roots samples showed protein (5.6 ±0.07)%, crude lipid (7.5 ±1.4)%, crude fibre (20.30 ±1.53)%, moisture (29.2 ±1.06)%, ash (8.00 ±1.56)%, and carbohydrate (58.60 ±0.01)%. Meanwhile the phytochemical screening analysis of the leaves of S. dulcificum showed the following concentrations in the leaf smples; alkaloids (0.6 ±0.20)%, saponins (2.80 ±0.20)%, flavonoids (2.8 ±0.2)%, polyphenols (3.52 ±0.10)% cardiac glycosides (3.44 ±0.20)%. Similarly, the root samples exhibited the following concentrations: saponins (0.6 ±0.02)%, polyphenols (4.30 ±0.10)%, anthraquinones (0.02 ±0.12)% and cardiac glycosides (4.40 ±0.20)%. On the other hand, the mineral elements concentrations of the leaf samples were as follows; Ca(0.001 ±0.00) mg/100 g, Cr(0.0006 ±0.00) mg/100 g, Fe(0.0029 ±0.01) mg/100 g, Zn (0.0095 ±0.00) mg/100 g Cu (0.00082 ±0.01) mg/100 g while that of root samples were recorded thus; Ca(0.00134 ±0.01) mg/100 g, Cr(0.00073 ±0.01) mg/100 g, ______

*Corresponding author: E-mail: [email protected];

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Zn(0.0097 ±0.01) mg/100 g Fe(0.00025 ±0.01) mg/100 g and Cu (0.007 ±0.01) mg/100 g. Generally, the leaf samples of S. dulcificum were rich in carbohydrate and moisture while the roots were rich in carbohydrate, moisture and fibre. Ash, fat and protein also showed remarkable concentration. Essential mineral elements were present at required concentration thus making S. dulcificum an important source of phytomedicine in the study area.

Keywords: Miracle fruit S. dulcificum; mineral content; chemical profiling.

1. INTRODUCTION of the tongue that causes every sour or acid foods eaten to taste very sweet. The test- Right from the creation, plants and its products modifying effect last up to two hours or more, derived from parts of plants such as stem bark, causing acid food substances such as sour lime, leaves, fruits and seeds have been parts of lemon, grape juice and even vinegar to taste phytomedicine, thus indicating that any part of a sweet [8]. plant may contain useful active compounds [1]. Medicinal plants constitute the main source of Chen [7] pointed out that the sweetness intensity raw pharmaceuticals and health care products is reduced by 0.02% citric acid after 0.4% [2]. Mandal [3] pointed out that extraction and purified miraculin solution is held in the mouth is characterization of several active phyto- equivalent to that of about 0.3% sucrose. The compounds from green plants have given birth to fruits are small approximately 2-3 cm long some high activity profile drugs. Edem and ellipsoid berries that are bright red when riped Dosunmu [4] also reported that plants are and composed of a thin layer of edible pulp primary sources of medicines, food, shelters and surrounding a single seed [9]. The leaves other items used by human every day. While are thin, papery, leathering and evergreen. Beroumand and Deokule [5] identified fruits as It is reported that African here used S. dulcificum sources of minerals, fibre and vitamins which to sweeten a sour gruel made from stale also provide essential nutrients for the human bread, soured palm win and pito, a sour health. alcoholic beverage made from fermented grain. It has also been used before eating a certain Thus, S. dulcificum has also been found as an type of sour corn bread [10-12]. The present important source of phytomedicine. S. dulcificum studies deals with the analysis of S. dulcificum is commonly known as miracle fruit. It is known obtained from Akwa Ibom State Nigeria with a as Mkpatun by the Ibibio and Efik people of Akwa view of obtaining it nutritional potentials and Ibom and Cross River States, Azimomo by the Therapeutic utility. Benin people of Edo State and Igbayum by the Yoruba people of Western Nigeria. S. dulcificum , 2. MATERIALS AND METHODS is a plant with a berry that when eaten, causes sour foods (such as lemons and limes) 2.1 Sample Collection and Preparation consumed to taste sweet [6]. The sweetening property is due to miraculin (a glycoprotein) The fresh leaf and root samples of S. dulcificum which is used commercially as sugar substitute. were collected from Ibot village in Mkpat Enin Apart from miracle fruit, it is also commonly Local Government Area of Akwa Ibom State. The known as miracle berry. But S. dulcificum should samples were identified in the Department of not be confused with two other plants species Botany University of Calabar, stored in a Gymnema sylvestre and Thaumatococcus polyethylene bag and dried at 160°C in an oven. daniellii referred to as miracle fruit which also After drying, both leaves and roots were blended affect perception of taste. to powder with a manual blender. The blended S. dulcificum (sapotaceae) is an ever samples were put in a desiccator and labeled green shrub native to tropical West Africa, accordingly and stored in a desiccator. and the fruits, and red berries have the property of modifying sour taste into sweet 2.2 Sample Extraction taste remarkably. The active material of the berry is the glycoprotein, miraculin, which has Petroleum ether and distilled water were used to no taste in itself [7]. The most outstanding extract each of leaf and root samples. In each property of the fruit is its effect on the taste buds process, 20 g of the powdered samples were

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washed and packed into extraction thimble and 2.7 Mineral Element Analysis fitted into soxhlet extractor. The samples were Soxhlet extracted for three (3) hours. The The methods of AOAC [14] were used in these petroleum ether and water extracts obtained determinations. Standard solutions were were put in the reagent bottles. Each bottle was prepared for each element. Sodium and distinctively labeled and kept in the laboratory for potassium were determined by flame photometer phytochemical screening. (Gallen Kamp). Calcium, magnesium, iron, chromium, lead, mercury, nickel, cadmium, 2.3 Digestion of Sample cobalt, manganese, zinc and copper, were determines using atomic absorption Five grams (5 g) of the powdered samples spectroscopy (AAS). were accurately weighed into a conical flask. 25 ml of concentrated nitric acid was added 3. RESULTS AND DISCUSSION to the sample. 5 ml of perchloric acid was then added to the sample. The conical flask Tables 1 and 2 show the results of proximate with its content was gently heated (50-70°C) compositions and phytochemical screening of the on a stuart hot plate until the colour changed leaves and roots of S. dulcificum respectively. from brown to colourless. The digest was made Similarly, Tables 3 and 4 show the results of up to 100 ml with deionized distilled water. quantitative determination of some Appropriate dilutions were made for each phytochemicals and mineral elements element. All determinations were carried out in compositions of leaves and roots of triplicates. S. dulcificum. The results of proximate analysis of S. dulcificum as given in Table 1 shows that 2.4 Proximate Analysis the moisture content of the leaves and roots of S. dulcificum are (40.30 ±1.53%) and Proximate analysis involves determination of (29.20 ±1.06%) respectively. The percentages moisture, ash, crude fibre, crude protein, fat and are slightly lower than the moisture content of carbohydrate contents [13]. Ash, moisture fat, Chrysophyllum africanum (66.67 ±0.02)% [4]. The fibre and crude protein were determined through moisture content of food is usually used as a methods prescribe by [14]. The percentage meaure of stability and susceptibility to microbial available carbohydrate (CHO) was calculated contamination [19]. These compositions indicate using expression that S. dulcificum can be stored for a long time without spoilage. The study on the ash content of % CHO = 100 – (% ash + % crude protein + the leaves and roots of S. dulcificum obtained % crude lipid + % crude fibre) were found to be (6.70 ±2.00%) and (8.00 ±1.56%) respectively. These compositions 2.5 Phytochemical Screening are higher when compared to (2.03%) reported by Edem [20] for Tetracarpidium conophorum. The phytochemical screening procedures carried Again the ash content of S. dulcificum was found out on the leaves and roots of S. dulcificium were to be nearly the same with Alpinia allighas adapted from the previous work on medicinal (6.40 ±0.15%) by Landan [21]. The results of plant analysis [15,16]. Alkaloids, saponins, the analysis for fibre contents as presented in flavonoids, cardiac glycosides, polyphenols, Table 1 are (17.50 ±0.50%) and (20.30 ±1.53%) phlobatannins, anthraquinones, anthranoids, for leaves and roots respectively. Comparing tannins and reducing compounds were screened. these values to that reported for Chrysophyllum africanum fruits (4.45 ±0.02%) by Edem and 2.6 Quantitative Determination of Some Dosunmu [4]. S. dulcificum has a very high Phytochemicals percentage of fibre. Emphasis has been placed on the importance of keeping fibre in takes low in Quantitative determination of the following phyto- the nutrition of infants and pre-school children constituents; alkaloids, saponins, flavonoids, [22]. cardiac glycosides, polyphenols, anthraquinones and tannins were determined using methods The crude lipid contents of the leaves and roots described by A.O.A.C [14], Sofowora [17], of S. dulcificum were found to be (12.0 ±2.00%) Obadoni and Ochuko [18] and Soladoye and and (7.50 ±1.40%) respectively. The lipid content Chukwuma, [1]. of the leaves of S. dulcificum is significantly

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higher than that reported for Chrysophyllum of the belly and collection of fluids in the body africanum (9.38 ±0.01) by Edem and Dosunmu [25]. The daily protein intake for children and [4]. Lipids are monosaturated and considered adult are 23-26 kg and 45-46 g respectively [26]. healthy when consumed on moderation. They However, the carbohydrate contents were found are essentials because they provides body with to be (57.18 ±0.01 and 58.60 ±0.01%) for leaves maximum energy [23]. Similarly, the protein and roots respectively. These values are higher contents were found to be (6.62 ±0.02%) and than (53.20%) reported for Tetracarpidium (5.60 ±0.07%) respectively. Protein is an conophorum fruit by Edem [19]. The result essential component of the diet needed for obtained in this study indicates that S. dulcificum survival of animals and humans. Basic function is is rich in carbohydrate. If carbohydrate is to supply adequate amount of required amino sufficient in food, it prevents the unnecessary acids [24]. Protein deficiency causes growth usage of protein and allows it to be used for the retardation, muscle washing, abnormal swelling body building processes.

Table 1. Proximate composition of leaves and roots of S. dulcificium

S/N Parameters Leaves (%) Roots (%) 1 Moisture 40.3 ±1.53 29.2 ±1.06 2 Ash 6.70.0 ±2.00 8.00 ±1.56 3 Crude lipid content 17.5 ±0.50 20.3 ±1.53 4 Crude fat content 12.0 ±2.00 7.5 ±1.40 5 Crude protein content 6.62 ±0.02 5.6 ±0.07 6 Carbohydrate 57.18 ±0.01 58.60 ±0.01 Data are: mean(x) ± standard deviation of triplicate determinations

Table 2. Phytochemical screening of leaves and roots of S. dulcificum

Bio-active compounds Petroleum ether Water extract Petroleum ether Water extract extract of leaves of leaves extract of roots of roots Alkaloid + - - - Saponnins: - + - ++ Flavonoids + - - - Polyphenols + ++ ++ ++ phlobatannins - - - - Anthranoids: - - - - Anthraquinones - - - + Cardiac glycosides + + ++ + Tannins + - - - Reducing compounds - - - - Key: + = present, ++ = present in large quantity, _ = not present

Table 3. Quantitative determination of some phytochemicals of leaves and roots of S. dulcificum

Bio-active compounds Leaves % Roots % Alkaloid 0.60 ±0.2 --- Saponnins: 2.80 ±0.2 0.62 ±0.02 Flavonoids 2.40 ±0.2 -- Polyphenols 3.52 ±0.01 3.44 ±0.01 Anthraquinones - 0.02 ±0.12 Cardiac glycosides 4.40 ±0.20 4.20 ±0.20 Tannins 4.00 ±0.20 - Data are: Mean (x) ± standard deviation of triplicate determinations

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Table 4. Mineral elements compositions of however higher than that of roots. The leaves and roots of S. dulcificum quantification of the leaves and roots revealed (2.8 ±0.2%) and (0.6 ±0.2%). Comparing the Element Concentration in Mg/100g levels of saponins in the leaves of S. dulcificum (Leaves) (Roots ) to that of Cissus populnea Guill and Perr. Calcium 0.001 ±0.00 0.00134 ±0.01 (Vitaceae). (2.85 ±0.35 mg/g) reported by Magnesium ND ND Soladoye [1]. This indicates that the leaves of Iron 0.0029 ±0.01 0.0025 ±0.01 S. dulcificum is very rich in saponins. The Potassium ND ND amount of saponins in S. dulcificum is, however Sodium ND ND lower than that of Chrysophyllum africanum Chromium 0.0006 ±0.00 0.00073 ±0.01 (3.663 ±0.02 mg/100 g) reported by Edem and Zinc 0.0095 ±0.00 0.0097 ±0.01 Dosunmu [4]. Manganese ND ND Lead ND ND It was found that the petroleum ether extracts of Copper 0.00082 ±0.01 0.007 ±0.01 the leaves contain a moderate concentration of Mercury ND ND flavonoids (Tables 2 and 3). Flavonoids were Nickel ND ND absent in water extracts of the leaves and roots. Cadmium ND ND Quantitative analysis showed the concentration Cobalt ND ND of (2.40 ±0.2%) flavoids. Thus, when compared to Key: ND = not detected, data are: mean (x) ± standard that of Cissus polulnea Guill and Perr (Vitacea) deviation of triplicate determinations (0.39 ±0.03%w/w) reported by Soladoye and Chukwuma [1], revealed that S. dulcificum is rich The phytochemical screening of the leaves and in flavonoids. Flavones are a class of flavonoids roots of S. dulcificum show that cardiac and are commonly attached to sugar to form glycosides were present in higher concentration glycosides. They are obtained from the synthesis in both petroleum ether and water extracts. of O-hydroxyacetophenone. O- Quantitatively, the levels of cardiac glycosides hydroxyacetophenone is benzoylated, followed were found to (4.40 ±0.2%) and (4.20 ±0.2%) by the base (pyridine/ KOH treatment of the respectively. These concentrations are higher benzoyl ether to effect an alkyl group migration to when compared to that of Cissus populnea Guill produce 1, 3- diketone [30]. and Perr. (Vitaceae ) (1.83)% reported by Soladoye and Chukwuma [1], cardiac glycosides Tannins showed a higher concentration in the have specific characteristics and powerful action petroleum ether extract of the leaves, but was exerted on cardiac muscles and therefore is used however absent in water extract of both roots in congestive heart failure due to dimination of and leaves. work capacity per unit weight of mycroches tissues, medicinal interest on cardiac glycosides Quantitative determination of tannins in the is because of their structure stimulant effect in leaves showed the levels of (40 ±0.20%). the heart [27]. Carnolides is a type of cardiac Sutharsingh [31] reported (8.72 ±0.44%w/w) for glycosides which is abundant in the Naravelia zeylanica Dc. This is lower than the asdepiadeceae and apynaceae. Their actions (40 ±0.20%) reported for S. dulcificum in the are anti-inflammatory, antiseptic and analgesic present investigation. and are used in the treatment of rheumatism [28]. Phytochemical screening result of reducing compounds in the leaves and roots of S. The result of phytochemical screening for dulcificum is given in Table 2. Reducing alkaloids show the presence of alkaloids in compounds were absent in both petroleum ether petroleum ether extracts. However, quantitative and water extracts of the leaf and root samples, analysis conducted on the leaves showed even after boiling for 30 minutes in a water bath. alkaloids concentration of (0.60 ±0.2%). This The result indicated that leaves and roots of quantity is lower when compared to the alkaloids S. dulcificum are not rich in reducing compounds content of Allium sativum (0.12 ±0.02) reported by compared to the seeds, which is evidence of the Huzaifa [29]. Screening result for saponins taste modifying activity. showed the presence of saponnins in water extract in both leaves and roots. The Phlobatannins were absent in both petroleum concentration of saponins in the leaves was ether and water extracts of the leaf and root

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samples. Similarly, the screening for anthranoids Zinc also recorded a low concentration in the showed the absent of anthranoids in both leaves and roots of S. dulcificum which were extracts (petroleum ether and water) of the leaf found to be (0.0095 ±0.00 mg/100 g) and and root samples (Table 2). Phytochemical (0.0097 ±0.01 mg/100 g) respectively. The screening analysis shows that the water extract concentrations are very low when compared to of the root shows the presence of antraquinones that of the seed of the same plant reported to be (Table 2) while the quantification of (2.710 ±0.009 mg/100 g) [37]. The zinc present in anthraquinones revealed (0.02 ±0.12%) (Table 3). the pancreas may aid in the storage of insulin, It was however, absent in the leaves sample. zinc in the plants could mean that plants can play Naturally occurring anthranol are easily oxidized essential roles in the management of diabetes, by atmospheric oxygen to anthraquinones. which result from insulin malfunction [42]. The Anthraceae are found as glycosides and results of the analysis for copper in the leaves aglycone [32]. Anthraquinones and related and roots of S. dulcificum is presented in Table glycosides are thatatus, which increased smooth 4. The result obtained showed that leaf contains muscles tone of the large intestine [33]. (0.00082 ±0.01 mg/100 g) while the roots contain (0.007 ±0.01 mg/100 g). Comparing the result to The mineral elements analysis of S. dulcificum that reported for Averrhoa bilimbil (0.07 ±0.01) showed the composition of calcium to be ppm [43], S. dulcificum is not a good sources of ± ± (0.001 0.00 mg/100 g) and (0.00134 0.01 copper. Mineral elements composition of the mg/100 g) for both leaves and roots respectively. leaves and roots of S. dulcificum for manganese These concentrations are very low when is given in Table 4. Manganese was not detected compared to that of calcium obtained from in both leaves and roots. But Jeremiah [37] ± Aspilla Africana leaves (1.04 0.03) mg/l [34] and reported manganese content of the seeds to be Achyranthes aspera Linn roots (849435) ppm (2.280 ±0.004 mg/100 g). [35]. About 200-400 g/day will provide the recommended daily calcium allowance of 360 Similarly, lead, mercury, cadmium, cobalt and mg-1200 mg for children and adults especially if Nickel were not detected in the samples but combined with other food stuff rich in calcium. Jeremiahs [37] reported that the seeds of Calcium is required in the body for normal growth S. dulcificum contain a Nickel concentration of of bones and teeth [36]. Mineral element analysis (0.240 ±0.028 mg/100 g). result for magnesium is given in Table 4. Magnesium was not detected in both the leaves 4. CONCLUSION and root of S. dulcificum . The mineral levels of potassium in the leaves and roots of The leaves of S. dulcificum are rich in S. dulcificum is signifincantly low as potassium carbohydrate and moisture. Similarly the roots was not detected (Table 4). But this is not so with are rich in carbohydrate, moisture and fibre. Ash, seed of the same plant the potassium in the seed fat and protein were appreciably present. is 569.500 ±2.820 mg/100 g [37]. Phytochemical screening of the extracts (petroleum ether and water) revealed that Table 4 shows a significant composition of iron in S. dulcificum is highly rich in cardiac glycosides both leaves and roots of S. dulcificum. The iron ± and polyhenols when compared to other concentrations were found to be (0.0029 0.01 detected bioactive compounds. From the ± mg/100 g) and 0.0025 0.01 mg/100 g for the quantitive analysis of the phytochemicals, leaves and roots respectively. The obtained tannins, cardiac glycosides, polyphenols, results are very low when compared to that flavonoids and saponins showed a high levels in reported for Securinegavirosa leaves (2.02%) the both leaves and roots. Elemental analysis [38]. The recommended intake of iron per day for revealed that all the detected mineral elements children and adult is 10-18 mg/day [26]. Iron is were present in a moderate concentration in both required for the formation of blood and its leaves and roots. deficiency causes anaemia [39]. Sodium was not detected in both leaves and roots of S. dulcificum ACKNOWLEDGEMENTS as presented in Table 4. Meanwhile, chromium composition of the leaves and roots of The authors appreciate the overwhelming ± S. dulcificum were found to be (0.0006 0.00 support of Umoh, Unyime Udoudo Department of mg/100 g) and (0.00073 ±0.01 mg/100 g) Pure and Applied Chemistry University of respectively. For most plants species Cr at Calabar, Calabar-Nigeria for responding to all 10 mg/kg levels is considered to be toxic [40,41]. correspondence related to this manuscript.

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